Influenza virus A (IFV) continues to be a significant public health problem worldwide. Between 20,000 and 40,000 deaths are attributed to IFV every year in the United States. Introduction of a new antigenic subtype either due to naturally occurring antigenic shift or by bioterrorism could lead to a pandemic with devastating consequences. The currently available killed and subunit IFV vaccines will have only limited success against pandemic strains of influenza. The next influenza pandemic could occur at any time and with little warning. The recent emergence of avian influenza H5N1, H7N7 and H9N2 viruses in humans has highlighted the ability of at least some avian subtypes to cross the species barrier into humans. Such events can cause pandemic outbreaks of influenza by the emergence of an avian-human reassortant virus with the ability to spread rapidly in a naive human population. Adenoviral vectors are known to activate innate immunity and can be administered via the mucosal or parenteral route. In general, adenoviral vectors are strong inducers of both humoral (including mucosal) and cell-mediated immunity (CMI). Under Specific Aim 1, adenoviral vectors expressing hemagglutinin (HA), nucleoprotein (NP), or matrix protein 2 (M2) from H5N1 or HA from H7N7 or H9N2 will be developed and evaluated for their ability to induce humoral and CMI responses and degree of protection to IFV challenge in a mouse model. NP and M2 proteins are relatively conserved among IFV subtypes and induce good cellular immunity, thus broadening the scope of immune responses against influenza. Defensins, small molecular weight proteins induced by toll-like receptor activation, can recruit inflammatory cells and amplify innate and adaptive immune responses. Immunization studies with purified HA of H5N 1 virus indicate that this HA is poorly immunogenic, therefore, the immunogenicity of HA of avian IFVs (H5N 1, H7N7 and H9N2) expressed by adenoviral vectors could be further enhanced by the use of adenoviral vectors expressing defensin. Under Specific Aim 2, the strategies for developing long-lasting and broad IFV immunity with a combination of adenoviral vectors expressing novel HA types or HA, NP, and M2 with or without defensin by sequential administration of nonhuman and human adenoviral vectors will be evaluated. Naive and primed inbred mice and outbred ferrets will be used for immunization and challenge trials to address the complex spectrum of immunity that would exist in humans. The successful completion of this project will advance our understanding towards the type of immunity and immunogen/s required for long-lasting and broad immunity for effective protection against pandemic IFV.